The first objective of this study is to isolate and characterize procollagen secreted by L929 fibroblasts in culture and to characterize procollagen isolated from subcellular fractions of these cells. The second objective is to isolate and characterize the active and inactive forms of prolyl hydroxylase from L929 cells and to compare these forms with the cell-free product obtained by translating the messenger RNA for this enzyme. We shall grow L929 cells in suspension on microcarriers of DEAE-Sephadex beads. This technique allows the production of large amounts of cells so that procollagen and prolyl hydroxylase can be isolated from a single source and subcellular fractions can be obtained. We will identify procollagen by labeling cells with 14C-amino acids and we will purify procollagen by ion exchange chromatography and gel filtration. We will purify active and inactive forms of prolyl hydroxylase by ion exchange chromatography, gel filtration and immunoprecipitation. We will translate the mRNA for prolyl hydroxylase using a ribonuclease treated reticulocyte lysate, isolate the product by immunoprecipitation and characterize the cell free product. We expect that such studies will contribute to an understanding of the mechanism which allows for the transcellular movement of procollagen while at the same time accounting for the selective retention of one of its post-translational modifying enzymes, prolyl hydroxylase.